Project Summary/Abstract All living cells display a complex array of sugar chains (glycans) in surface and extracellular compartments. Vertebrate glycans often terminate with a family of 9-carbon backbone sugars called Sialic Acids (Sias), attached in various linkages, and modified in multiple ways. The long-term theme of this grant has been to unravel mechanisms generating this diversity, elucidating roles in health and disease. Over 30+ years we focused on Sia linkages, O-acetylation of the side chain, and most recently, human evolutionary loss of N-glycolylneuraminic acid (Neu5Gc). Trace amounts of Neu5Gc found in human epithelia and endothelia were explained by metabolic incorporation from foods, primarily red meats??the first example of a xenoautoantigen. Incorporation by commensal H. influenzae likely triggers polyclonal xenoautoantibodies?, which interact with Neu5Gc on human cells, causing ?Xenosialitis, an inflammatory response that aggravates progression of cancers and atherosclerosis in human-like Neu5Gc-deficient mice??likely relevant to human disease risks associated with red meat. Further complexity arises because some prokaryotes express ancestral nonulosonic acids (NulOs), and/or utilize convergent evolutionary mechanisms for pathogen molecular mimicry of vertebrate Sias. Many Sia/NulO modifications of potential biological and pathological significance remain largely unexplored, and few labs address this technically difficult area. Indeed, many approaches underestimate or even completely miss this biologically important diversity. Instead of logical next steps arising from recent successes, our new aims are focused on delivering maximum value for the future, leveraging knowledge from years of NIGMS support. We will complete Neu5Gc mouse model and population studies and attempts to characterize human monoclonal antibodies against Neu5Gc-glycans, but then spin off successful results to other investigators. We will then undertake risky explorations of aspects of Sia/NulO diversity that no other group is pursuing in-depth at this time. Attention will be on potential metabolic incorporation into glycoconjugates of human and mouse cells and into certain bacteria, and on analysis of circulating antibodies directed against NulO-glycans. We will study 4-O- acetylation, a modification so far not found in humans, defining it either as a human modification affecting biological processes, or a second example of a non-human xeno-autoantigen from food sources. We will similarly study legionaminic acid, a bacterial NulO with the same stereochemistry as human Sia. Small quantities incorporated into human tissues could trigger inflammation, via circulating antibodies against Leg-glycans. We will also study Kdn, a Sia thought not to be expressed on mammalian glycans, and yet derived from mannose in human and murine cells. Evolutionary conservation of Kdn production may help buffer mannose levels. However, Kdn may be expressed in small amounts on human cells under certain conditions, despite circulating antibodies. Each new aim addresses poorly studied yet common Sia/NulO, each with similarities and differences from Neu5Gc. We anticipate many novel findings. While exploratory, some studies address defined hypotheses.